Modular compositing-multiple lot screening protocols for detection of pathogens, microbial contaminants and/or constituents

ABSTRACT

Provided are methods for sampling, testing and validating test lots, comprising: assembling a plurality of product portions from each of a plurality of test lots and combining the portions to provide a corresponding set of test lot samples; enriching the test lot samples; removing portions of each enriched sample, and combining the removed portions to provide a modular composite sample; and testing of the modular composite sample, and individual testing of the enriched test lot samples, using at least one suitable detection assay for a target microbe or organism, wherein when such testing is negative all test lots are validated, and wherein when such testing is positive with respect to the modular composite sample, or with respect to an individual enriched test lot sample, individual test lots may nonetheless yet be validated by further testing of a portion of respective initially-negative enriched test lot samples and obtaining negative results.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Provisional patentapplication Ser. No. 11/199,871, filed 8 Aug. 2005, which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.60/599,473, filed 6 Aug. 2004 and entitled MODULAR WETCOMPOSITING-MULTIPLE LOT SCREENING PROTOCOLS FOR DETECTION OF PATHOGENS,both which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

Aspects of the present invention relate generally to improving theefficiency of producing food and health related (e.g., pharmaceutical)products, and the safety and quality thereof. Particular embodimentsrelate to novel methods for microbial pathogen testing that allow forenhanced sensitivity, cost savings and traceability (e.g. sourcetracking) for remedial purposes. The methods are broadly applicable tomany product areas including, but not limited to beef, pork, sheep,bison, deer, elk, poultry (e.g., chicken and turkey) and fish and otherseafoods, produce, juices, dairy products, dry goods (cereals, nuts,etc), fruits and vegetables, herbs and spices, and all manners of rawand processed foods, environmental samples (water, wastewater, soil,surface samples, air samples taken by impingers and filtration, etc),pharmaceuticals, and other types of samples to be analyzed usingmicroorganism enrichment-detection protocols.

BACKGROUND

Manufacturers and respective regulatory agencies strive to continuallyimprove the safety and the quality of foods, pharmaceuticals,neutraceuticals, water and the environment. Under recent United StatesDepartment of Agriculture, Food Safety and Inspection Service (FSIS)directives and guidelines, many food producers in the United States haveadopted sampling plans that involve testing, for example, of raw beeftrims and/or ground beef for E. coli O157:H7, and ready-to-eat productsfor Listeria monocytogenes and/or Salmonella. Similar trends areobserved in other countries, for products detained for domesticconsumption and/or export. For example, the pharmaceutical industryspends considerable resources on sterility testing and environmentalmonitoring, and the water and wastewater industry and their respectiveregulatory agencies allocate considerable resources to monitor thequality of water (e.g., drinking, recreational and fisheries resources),wastewater (treated and untreated) and sludge (biosolids). Concern forthe quality of air has resulted in monitoring of indoor air for thepresence of microbial contaminants and pathogens. These sampling plansassociated with such testing determine, for example, the disposition ofproducts and the production lots, or result in decisions regarding thesafety of water, receiving waters and recreational waters, and theimpact of wastewater discharge, and quality of air.

The operative art with respect to such product “hold and release”monitoring efforts comprises various different sampling plans, typicallypursuant to International Commission on Microbial Specifications forFood (ICMSF) guidelines for testing food products, FDA and USDA or EPAguidelines, or intuitive sampling plans. The common feature of thesesampling plans is that several samples representing production lots, ora number of environmental samples, are combined into a combinedproduction lot prior to sampling. For example, a typical plan formicrobial pathogen detection calls for taking more than one sample, forinstance ten (10) to sixty (60) sample pieces from a given sample lot(combined production lot), to form a composite sample for testing. Thenumber of pieces taken to form the composite sample depends, under ICMSFor other regulatory guidelines, on the outcome of infection/poisoning(e.g., the severity), the level of hazards, and potential increase inthe hazard levels due to storage. The same holds true in detectingmicrobial constituents and/or spoilage organisms. With respect to E.coli O157 testing, for example, a typical plan calls for taking 60samples (e.g., sample pieces) from a production lot to form a singlecomposite sample. With respect to Listeria monocytogenes, thirty (30)samples are typically taken from a production lot to form a compositesample. With respect to sterility testing, a typical plan may call forcompositing 10-100 units of the product, and in the case ofenvironmental sampling, 2-10 samples are often composited. Generally,the number of samples that are composited reflects: the overallsensitivity of the test method and the limit of detection that appliesto the test unit; the ability to concentrate/enrich the composite sampleto achieve the desired sensitivity; and the cost of sampling comparedwith the cost of analysis.

The costs associated with testing food are substantial, and consequentlymanufacturer's often resort to increasing the size of the “productionlot” to be tested. For instance, the standard size production lot fortrim testing for E. coli O157 in the U.S. beef industry is five (5)‘combos’ (each combo weighing approximately 2,000 pounds), and forListeria and Salmonella in ready-to-eat (RTE) products, it is onecomposite sample per production line per shift. A primary problemassociated with the use of large size test lots is that a large quantityof products must be downgraded or destroyed when there is a positivefinding of a pathogen. Additionally, because of the nature ofcombination ‘lotting’ (combining, for purposes of forming the test lot,several independent sub-lots that collectively span a long period ofproduction time), it is very difficult to investigate the cause of thefailure and pinpoint the source/cause, and remedial measures cannot beeffectively taken. Furthermore, compositing a large number of samplesmay result in reduced sensitivity for the test unit and adversely effectthe limit of detection.

Typically, once a composite sample is constructed (following the artwith respect to particular testing) it is then enriched for the presenceof a given pathogen/microbe by addition of appropriate amounts ofenrichment media and incubation at an appropriate temperature for agiven amount of time. If the pathogen/microbe is present, then it growsand multiplies under the favorable conditions of enrichment, therebyproviding more material which can be detected by subsequent analysis.The composite sample is then tested by one or more available, artrecognized methods including, for example, enrichment followed byimmunoassay-based tests or PCR-based methods, or culturing of theorganism of interest, or other DNA- or immunochemical-based methods.

The above-described prior art has several substantial deficiencies.First, even though the prior art production lot encompasses severalproduction sublots (for instance 5 combo/pallets of products, or anentire shift of production), if a positive finding is obtained for theproduction lot (composite test sample), the entire production lot isrejected (all 5 combos/pallets, or one shift of production) and divertedto economically undesirable end uses (e.g., cooking or disposal), eventhough the pathogen of concern may be confined to only a limited portionof the production sublots (corresponding to one or two combos/pallets oran hour of production) comprising the production lot.

Second, the current art does not allow for retesting of any productionsublots, primarily because microbial contaminants are unevenly spreadthroughout the products, and in many instances the levels ofcontamination are minute and may be present on very small portions ofthe products in each sublot. Therefore, once a composite sample isconstructed if it tests positive all of the sublots are destroyed. Theregulatory agencies do not allow for re-testing of the sublots, premisedon the argument that after a production lot tests positive, negativetest results obtained for production sublots, or even a new productionlot composite sample, are meaningless since the microbial contaminantsare not uniformly present throughout the products.

Third, the current protocols for collecting a composite samplerepresenting a production lot often group together production sublotsthat are unrelated by virtue of product type or hours of production.This makes investigating the nature of the failure a difficult task evenwhen relevant information is available. As an example, information isfrequently available on a ‘per combo’ basis including (but not limitedto) hour of production, vendor source of raw materials, productionemployees present, and operational status of microbial interventionprocess steps. However, when five unrelated combos/pallets are includedin a production lot it is much harder to rationally analyze theavailable information since there is no way to determine which combo(s)contained the pathogen (as stated above, prior art re-sampling is not aviable option since it may not yield the same result). Likewise, forsterility testing, when the composite sample tests positive, the entireproduction will be disposed.

Therefore, there is a pronounced need in the art to implement novel andeffective testing protocols that provide a greater measure of assurancethat the food product is safe, that provide economic relief to theproducer, and that allow for effective tracking of the contaminatedlot(s) for remedial purposes (e.g., pinpointing the time of thecontamination and determine the segment of production which wasimpacted).

SUMMARY OF THE INVENTION

Aspects of the present invention provide novel enrichment, testing anddetection methods for detection of pathogens or other microbes in, forexample, food, water, wastewater, industrial, pharmaceutical, botanical,environmental samples and other types of samples analyzed by detectionmethods (e.g., enrichment-detection methods). According to particularaspects, where any microbiological testing program involves compositinga number of samples to form a test lot, independent enrichment of eachof the components of the composite sample is performed with subsequentformation of a modular (e.g., wet modula) composite sample by combiningportions from each independent enrichment, which allows for determiningthe outcome of the test for each individual subunit of the composite asopposed to generalizing the results of the test sample to all of it'ssubunits. This individual outcome determination is achieved by retestingthe individual enrichment samples in the event that the compositeenrichment tests positive.

In preferred aspects, a reportable lot is defined as a single unit of amodular (e.g., modular wet) composite lot, rather than as an entirecomposite lot comprised of multiple sublots. For example, a reportableproduction lot is defined as a single unit of production and correspondsto a single unit of a modular wet composite production lot, rather than(as in the prior art) as an entire composite lot comprised of multipleproduction units (multiple sublots). With respect to testing eachreportable lot (e.g., each single unit of production), a plurality ofpieces/portions of sample are separately collected from each singlereportable lot (e.g., from each single production unit; individual testunit) and composited to form, for example, one ‘single-unit productionlot sample’ (corresponding, in each case, to a particular single unit ofproduction), which is enriched using appropriate enrichment protocols toallow for the levels of the target organism to reach detection levelsand uniformity of presence in the enriched sample. Multiple aliquots ofthe enrichment media may be removed for analysis, thus circumventing theprior art problem of having to re-sample a test production lot wheremicrobial contaminants are not uniformly present throughout the product.

In the exemplary context of enriched single units of production, equalportions of enrichment buffer are removed (aseptically) from eachsingle-unit production lot sample and combined to form a Modular WetComposite Sample (MWCS). The MWCS is then tested using an appropriatetest (detection assay) for the target agent (e.g., target organism). Ifa negative result is obtained from the MWCS, then all of the individualsingle-unit production lots are released. Significantly, if a positiveresult is obtained from a MWCS, the enrichment buffers from each of theindividual single-unit production lot samples are separately(individually) tested, using the same test protocol, or a test protocolwith enhanced sensitivity, or any appropriate test protocol. Accordingto particular preferred aspects, the signal obtained from a MWCS is dueto one or more of it's components (e.g., reportable lots (e.g.,individually enriched single production units)), and it is, therefore,possible in all cases to trace a positive signal to one or moreindividual subunits by testing each individual enrichment sample for thesame target. When a positive finding is obtained for any individualreportable lot sample (e.g., single-unit production lot sample), thatparticular reportable lot (e.g., single-unit production lot) is deemedto be positive, and appropriate actions are taken based on the result.In the case of pathogen/microbial testing for foods, the individualpositive sublots can be diverted to cooking, disposal, or otheracceptably safe endpoint. The rest of the individually enriched testunits that test negative are deemed to be negative and will be reportedas such (are validated).

DETAILED DESCRIPTION OF THE INVENTION

Particular aspects provide novel enrichment, testing and validationmethods for detection of pathogens or other microbes in any type ofsamples (e.g., food production lots, water, wastewater, industrial,pharmaceutical, botanical, environmental samples, etc.) analyzable byenrichment-detection methods. In preferred aspects, a test lot(single-unit production lot), rather than (as in the prior art) acomposite lot comprised of multiple test lots (e.g., single-unitproduction lot units), is defined and established as the single unit ofproduction. A plurality of portions (e.g., product portions) areseparately collected from each test lot (e.g., single-unit productionlot) and composited to form a corresponding set of test lot samples(e.g., single-unit production lot samples), which are enriched usingsuitable enrichment protocols to allow target agents/organisms to reachdetectable levels and sample uniformity. In particular embodiments,equal portions of enrichment buffer are removed (preferably aseptically)from the enriched samples, and combined to form a modular compositesample (e.g., modular wet composite sample; MWCS). The MWCS, forexample, is tested using a suitable detection assay for the targetagent/organism. If a negative result is obtained from the MWCS, then allof the individual single-unit production lots are validated andreleasable. If a positive result is obtained from a MWCS, the enrichmentsample corresponding to individual test lots (e.g., individualsingle-unit production lots) are separately (individually) tested, usingthe same, or a more sensitive test protocol. If a positive finding isobtained for any test lot sample (e.g., individual single-unitproduction lot sample), that particular respective lot is diverted tocooking, disposal, or other acceptably safe endpoint. If a negativefinding is obtained for any individual test lot sample (e.g.,single-unit production lot sample), then that particular lot isvalidated and releasable. In this manner, any member lot of the MWCSthat tests negative using the same method (about 5-fold more sensitiverelative to the prior art), or an enhanced method (about fifty timesmore sensitive), will be validated and releasable for consumption.

DEFINITIONS

The term “test lot” or “reportable lot” as used herein to refer to anassemblage of one or more specimens of a medium or process (e.g.,assemblage of specimens of air, water, solids, or of products of aproduction process, etc.), where such assemblage can be sampled bytaking portions of the one or more specimens thereof, and where the oneor more specimens of the assemblage are operationally linked in a manner(e.g., proximity, time, process step, etc.) whereby information derivedabout sampled portions is operationally applicable to all specimens ofthe assemblage, and thus to the test lot. In particular instances,portions are synonymous with specimens.

The term “single production unit” or “single-unit production lot” asused herein is a form of test lot, and refers to a single unit ofproduction (e.g. for beef trim a “combo,” or for almonds a “bin”) ratherthan, as in the prior art, to a composite comprised of multiple,non-operationally linked single production units (production sublots).

The term “product portion” as used herein refers to a product piece(e.g., a piece of solid beef trim, etc.), aliquot of product (e.g., avolume of liquid juice) or weight of product (e.g., a weight ofsemi-solid pudding). A single unit of production is composed of multipleproduct portions. In preferred embodiments, the number of productportions which comprise a single unit of production is great enough thatremoval of some product portions to form a sample does not impose anunacceptable economic loss to the producer. In particular instances, aproduct portion may be a specimen of a test lot as defined above.

The term “compositing” as used herein refers to combining a number ofsingle product portions to form one larger sample. In preferredembodiments, the number and selection of single product portions to becombined conform to statistical-based sampling plans which seek to forma typical or average sample that is representative of the test lot(e.g., single production unit) being sampled.

The term “test lot sample” or “single-unit production lot sample” or“single production unit lot sample,” as used herein refers to a sampleformed by combining multiple product portions from a test lot (e.g.,single production unit), such that each test sample (e.g., eachsingle-unit production lot sample) is attributed to a particularcorresponding test lot (e.g., production unit). For production lotsamples, the number of portions/pieces combined many be essentially anynumber, but preferably is selected so as to conform to statistical-basedsampling plans. Preferably the number of pieces combined is selectedfrom the range group consisting of from about 5 to about 100, from about10 to about 90, from about 20 to about 80, from about 30 to about 70,from about 30 to about 60, and from about 40 to about 50 productportions. Preferably, the number of independent product portions isselected so as to yield a sample size that is equal to or greater thanthat required by the subsequent analysis to be applied.

The term “enrichment” as used herein refers to incubating a test lotsample (e.g., single-unit production lot sample) under conditionssuitable to allow levels of a target agent/organism that is present toreach detectable levels and become uniform or substantially uniformthroughout the enriched test lot sample (e.g., single-unit productionlot sample). Preferably, enrichment comprises addition of an amount ofan enrichment medium or buffer to a sample, and incubating the sample ata favorable temperature for a period of time sufficient to allow theorganism to grow and multiply.

The term “enrichment medium” as used herein refers to a mixture whichcontains nutrients and which has properties (e.g., pH and/or oxygencontent) which favor the growth of the target agent/organism.Preferably, the enrichment medium is formulated to mimic the environmentof the target agent/organism or take advantage of a known metabolicproperty of the target agent/organism. Preferably, the enrichment mediumwill omit nutrients needed by competing undesired agents/organisms.Preferably, the enrichment medium will include selective agents thatwill inhibit competing undesired agents/organisms. An example of anenrichment medium is that used for the isolation of nitrogen fixingbacteria (those that can use nitrogen gas as their sole nitrogen source)contains no source of fixed nitrogen in the medium. A second example isan enrichment medium for the isolation of bacteria capable of utilizing2,4-dichlorophenoxyacetic acid (also know as 2,4-D, a common herbicide)as a source of carbon and energy. The enrichment medium is formulated tocontain benzoate as the only organic compound.

The term “wet enrichment” as used herein refers to diluting a solid,semisolid or liquid single-unit production lot sample (single productionunit lot sample) with enrichment medium in a ratio of approximately 1:10(wt./v), consistent with conventional methods for expanding and testinga variety of target agents/organisms as set forth in widely recognizedpublished methods. For example, the conventional method for expandingand testing for coliforms, fecal coliforms and E. Coli in food,comprises a 1:10 dilution of the samples (e.g., 50 g into 450 ml) (see,e.g., U.S. FDA Bacteriology Analytical Manual Online, Chapter 4 and 4A,describing standard 1:10 dilution procedures for testing of coliforms,fecal coliforms and E. Coli in food, shellfish and juices).

The term “dry enrichment” as used herein refers to enriching a solid,semisolid or liquid single unit production lot sample either with noaddition of other compounds if the sample's intrinsic properties (wateractivity, nutrients and pH) are sufficient/proper to promote the growthof the target agent/organism, or by dilution of the sample with minimalamounts of enrichment medium in ratios ranging from about 0.1 (wt/v) toabout 1 (wt/v).

The term “modular composite sample” as used herein refers to a sampleformed by removing, preferably aseptically, equal portions of each of aplurality of enriched single-unit production lot samples, and combiningthe removed portions.

The phrase “suitable detection assay” as used herein refers to any assaythat is suitable for detecting a particular agent/organism. Preferably,the assay is optimized to detect the particular agent/organism, and maybe combined with one or more concentration steps for concentration ofthe agent/organism being assayed.

The term “validation” as used herein with respect to production lotsrefers to determining that a particular sample tests negative using thedetection assay. Where a modular composite sample tests negative, allsingle-unit production lot samples comprising the composite sample arevalidated along with the respective single-unit production lots, whichare thus cleared for release. Where a modular composite sample testspositive, individual single unit production lots may nonetheless yet bevalidated if particular respective single-unit production lot samplestest negative.

“Trim” refers to small pieces of meat and fat which are excised during,for example, the beef fabrication process in order to produce primal andsubprimal pieces and marketable cuts.

“Trim testing” refers to the process of testing trim, or raw materialswhich are to be used for ground meat production for microbial/pathogencontent.

A “combo” or “combo-bin” refers, in the beef industry, to the singleproduction unit packaging unit. Alternatively raw materialscorresponding to a single production unit to be tested can be packagedinto boxes, bags or other appropriate containers, which can be placed,for example, on pallets.

A “composite lot-unit” or “five-combo-lot unit” refers to a compositeunit, comprised of five combos (combo-bins). In prior art samplingplans, the composite lot-unit represents the raw material (compositetrim) upon which sampling, testing and acceptance or rejection is based.

Therefore, according to particular aspects, a test lot (or test unit)(e.g., a single-unit production lot or single production unit lot) isdefined and taken as one or more operationally-linked specimens of amedium or process. A test lot may be a single unit of production (e.g.,a single combo, bin, pallet, or segment of production, etc.), or asingle environmental sample (e.g., single air sample, single water andwastewater sample, etc.), rather than, as in the prior art, as acomposite of multiple production units or environmental samples.

In preferred embodiments, a number of pieces or portions of sample arecollected from a test lot (e.g., from a single production unit (e.g.,from the single combo, bin, pallet, or segment of production, etc.), andcomposited to form one ‘single-unit test sample’ (or ‘single-unitproduction lot sample.’ In particular embodiments (e.g., wetenrichment), enrichment buffer is added to the sample. In alternateembodiments, ‘dry enrichment’ (as defined herein above) is done.

Significantly, in the inventive modular (e.g., wet modular) compositingmethod, samples taken from each unit of production are enrichedseparately using appropriate enrichment protocols to allow for thelevels of the target organism to reach detection levels and uniformityof presence in the enriched sample. In preferred aspects, suchenrichment obviates the prior art problem of having microbialcontaminants that are not uniformly present throughout the productsample. Specifically, while microbes are not necessarily uniformlydistributed in food, water, air, pharmaceuticals, etc, after enriching asample as described herein, the target microbes are uniformly, or asleast substantially uniformly, present in the enrichment medium orextract from dry or semi dried enrichments.

Following incubation for the target organism, equal portions ofenrichment buffer or sample are removed (aseptically) from each ofseveral test lot samples (e.g., from several single-unit production lotsamples; preferably 5) and pulled together to form a Modular WetComposite Sample (MWCS). The MWCS is then tested using an appropriatetest (detection assay) for the target organism. If a negative result isobtained from the MWCS, then all of the individual single unitproduction Lots are released.

If a positive result is obtained from a MWCS, the enrichment buffersfrom each of the individual single unit production lot samples are thenseparately (individually) tested, using the same test protocol, or anyother appropriate tests. Alternately, an augmented protocol that uses aconcentration step for the target organism, such as immunomagnetic beadseparation, affinity chromatography, etc., followed by an appropriatedetection method is used to test the enriched individual test lotsamples (e.g., the individual single-unit production lot samples).

In preferred aspects, the inventive methods of analysis are modified byintroducing a concentration step (e.g., employing antibody-coatedparamagnetic beads into the procedure (e.g., DNA-based detection,biosensor based detection, classical microbiological detections, etc)).Antibody-coated paramagnetic beads, for example, afford an approximately10-fold increase in sensitivity. This concentration step, combined withthe fact that the individual test lot samples (e.g., individualsingle-unit production lot samples) are not 5-fold diluted relative tothe MWCS, allows for an overall 50-fold increase in sensitivity relativeto prior art sampling and testing protocols. If a lateral flow device isused for detection, such immunomagnetic concentration may or may not beused, in which case the detection sensitivity is increased by about5-fold.

Where a positive finding (test result) is obtained for any individualtest lot sample (e.g., single-unit production lot sample), thatparticular corresponding test lot (e.g., single-unit production lot) isdeemed to be positive, and appropriate decisions are then made on thepositive results (e.g., in the case of food products, diversion tocooking, disposal, or other acceptably safe endpoint). For each of therest of the test lot samples (e.g., single-unit production lot samples)belonging to the positive MWCS, where a negative finding is obtained,that particular test lot sample (e.g., that single-unit production lotssample) is deemed to be negative. In this manner, any test lot (e.g.,any single-unit production lot) that is a member of the group of testlots (e.g., single-unit production lots) that comprise a correspondingMWCS, will be considered to be negative, if it tests negative using thesame method (about 5-fold more sensitive relative to the prior art), orthe enhanced method (about fifty times more sensitive).

In alternate preferred embodiments, a new Modular Wet Composite Sampleis reconstructed from the member lots (test lots) that have testednegative, and subjected to a third layer of testing to again show(confirm) that they are negative. Specifically if one of the fiveindividual test lots (e.g., individual single-unit production lots) hastested positive (and the other four negative), and the protocols callfor testing of composites consisting of five test lots, a new singletest lot sample can formed from a new test lot, and can be used alongwith the four negative samples to form a new MWCS.

The methods of the present invention provide substantial economicsavings to the food and pharmaceutical industries, because of theprecision in detecting in the context of operationally-linked portionsof production (e.g., test lots; single production units; single-unitproduction lots) which are contaminated. The inventive methods preventunnecessary destruction of negative lots that are condemned because ofguilt by association (e.g., simply because they were grouped with acontaminated composite prior art multiple production unit lot in a priorart testing protocol). Where a positive finding is obtained, a smallerquantity of material is diverted to an economically undesirable end use.The proposed invention has substantial utility (e.g., bringing savings,accuracy and precision in pinpointing problems) in the environmentalindustry.

Additionally, and significantly, it is easier to trace the source of thecontamination and apply remedial measures, because the single-unit testcomponent points, for example, to single point of production or a singlesampling site.

Particular Preferred Aspects:

Particular aspect provide a method of sampling, testing and validatingtest lots, comprising: separately collecting a plurality of portionsfrom each of a plurality of test lots, the test lots each comprising anassemblage of one or more operationally-linked specimens, wherein theassemblage can be sampled by taking portions thereof; combining thecollected portions corresponding to each of the test lots to provide acorresponding set of test lot samples, wherein each test lot sample isattributed to a particular corresponding test lot; incubating the set oftest lot samples under conditions suitable to allow levels of a targetagent or organism that is present in one or more of the samples to reachdetectable levels and become uniform, or substantially uniform,throughout the respective one or more samples, to provide a set ofenriched test lot samples; removing, aseptically, equal portions of eachenriched test lot sample, and combining the removed portions to providea modular composite sample; and testing of the modular composite sample,using a suitable detection assay, for the target agent or organism,wherein where such testing is negative all test lot samples arevalidated, and wherein where such testing is positive, individual testlots may nonetheless yet be validated by further testing of a portion ofthe respective enriched test lot sample using the same or a moresensitive protocol and obtaining a negative test result.

In particular aspects, the test lot is selected from the groupconsisting of an environmental lot comprising one or moreoperationally-linked environmental specimens, pharmaceutical lotcomprising one or more operationally-linked pharmaceutical specimens,single-unit product lot comprising one or more operationally-linkedproduct specimens, and combinations thereof. In particular aspects, thetest lot is a single-unit production lot, comprised ofoperationally-linked product specimens. In particular aspects, productspecimens are synonymous with product portions. In particular aspects,the methods further comprise incubating the modular composite sampleprior to testing it. In particular aspects, incubating each test lotsample comprises adding an amount of enrichment medium to the sample,and removing enriched sample portions comprises removing a portion ofthe enrichment medium from each enriched sample and combining theremoved portions to provide a modular composite sample. In particularaspects, testing of the modular composite sample, using a suitabledetection assay comprises use of a concentration step to concentrate themicrobial agent or organism.

In particular aspects, where testing of the modular composite sample ispositive, and wherein the individual test lots are further tested, suchfurther testing comprises use of the more sensitive protocol.Preferably, the more sensitive protocol comprises use of a concentrationstep to concentrate the microbial agent or organism, in combination withthe same test protocol used to test the modular composite sample.

In particular aspects, the methods further comprise forming anadditional modular composite sample from the enriched test lot samplesand testing the additional modular composite sample, using the same or adifferent suitable assay, to confirm the testing status. In particularaspects, the methods further comprise forming an additional modularcomposite sample from an additional enriched test lot sample along withany enriched test lot samples that test negative, and testing theadditional modular composite sample, using the same or a differentsuitable assay. In particular aspects, the methods further comprise,where testing of the modular composite sample is negative, forming anadditional modular composite sample from the enriched test lot samples,and testing the additional modular composite sample, using a differentsuitable assay, to confirm the negative testing status.

In particular aspects, the test lot is selected from the lot groupconsisting of a single combo, single bin, single pallet, and a segmentof production. Preferably, the number of test lots is from about 3 toabout 10, about 5 to about 8, or about 5.

In particular aspects, the methods further comprise, where testing ofthe modular composite sample is positive, determining which individualtest lots are positive, and application of remedial measures that arespecific to one or more individual test lots that test positive. Inparticular aspects, separately collecting a plurality of portions fromeach of a plurality of test lots comprises separately collecting fromeach test lot a number of portions selected from the group consisting offrom about 5 to about 100, from about 10 to about 90, from about 20 toabout 80, from about 30 to about 70, from about 30 to about 60, and fromabout 40 to about 50 portions. Preferably, separately collecting aplurality of portions from each of a plurality of test lots comprisesseparately collecting from each test lot about 30 to about 60 portions.Alternatively, separately collecting a plurality of portions from eachof a plurality of test lots comprises separately collecting from eachtest lot about 30 portions. Preferably, separately collecting aplurality of portions from each of a plurality of test lots comprisesseparately collecting from each test lot about 60 portions.

Particular aspects provide a method of sampling, testing and validatingtest lots, comprising: separately collecting a plurality of portionsfrom each of a plurality of single-unit production lots, the single-unitproduction lots each comprising an assemblage of one or more productspecimens, wherein the assemblage can be sampled by taking portionsthereof; combining the collected product portions corresponding to eachof the single-unit production lots to provide a corresponding set ofsingle-unit production lot samples, wherein each single-unit productionlot sample is attributed to a particular corresponding single-unitproduction lot; incubating the set of single-unit production lot samplesunder conditions suitable to allow levels of a target agent or organismthat is present in one or more of the single-unit production lot samplesto reach detectable levels and become uniform, or substantially uniform,throughout the respective one or more samples, to provide a set ofenriched single-unit production lot samples; removing, aseptically,equal portions of each enriched single-unit production lot sample, andcombining the removed portions to provide a modular composite sample;and testing of the modular composite sample, using a suitable detectionassay, for the target agent or organism, wherein where such testing isnegative all single-unit production lot samples are validated, andwherein where such testing is positive, individual single-unitproduction lots may nonetheless yet be validated by further testing of aportion of the respective enriched single-unit production lot sampleusing the same or a more sensitive protocol and obtaining a negativetest result.

The following Examples are exemplary are not intended, and should not beconstrued to limit the scope of the aspects of the present conceptionthat are claimed hereunder.

Example I The Present Inventive Methods were Shown to Provide aSubstantial Economic Advantage, while Providing for Enhanced Safety, andImplementation of Remedial Measures

At present, the standard size ‘production lot’ for trim testing for E.coli O157 in the beef industry in the USA is a combination of fivecombos, where each combo represents a single production unit and weighsapproximately 2,000 pounds. An inventive modular composite samplemethod, and in particular a Modular Wet Composite Sample (MWCS) methodwas implemented at a United States beef producer. TABLE 1 shows,according particular aspects, the result of testing 62,919 MWCS samples,and then retesting the individual single-unit production lot (combo)samples whenever a positive result was obtained from the MWCS sample.

TABLE 1 shows that out of 62,919 MWCS samples, a total of 217 yielded apositive testing result for E. coli O157. Using prior art protocols, allof the single-unit production lots that comprised the 271 positivesamples would go to rendering or be downgraded for cooking. As each MWCSrepresents five single-unit production lots of 2,000 lbs each, thiswould represent approximately 2,710,000 pounds of product (i.e.,271×5×2,000 pounds=2,710,000 pounds).

Specifically, the individual single-unit production lot samplescomprising the MWCS were analyzed separately following each positiveMWCS result. As can be seen in TABLE I, only 391 individual single-unitproduction lot samples yielded a positive result out of the cumulativetotal of 1,085 individual single-unit production lot samples comprisingthe MWCS samples which yielded a positive result. If only the 391individual single-unit production lots yielding a positive sample resultwere diverted to rendering or downgraded for cooking this wouldrepresent 782,000 lbs of product. The remaining 694 individualsingle-unit production lots, corresponding to 1,928,000 lbs of product,are, according to preferred aspects of the present invention, validatedand releasable into the chain of commerce.

The product, in this Example I, being produced by this U.S. beefproducer was beef trim. The release of the validated 19,280,000 lbs ofbeef trim into commerce using the present invention, instead ofdiverting or downgrading the product, trim, represents a cost savings tothe producer of about $2,500,000, relative to prior art methods.Additionally, the testing of the single-unit production lots byaugmented testing protocols (representing a 5- to 50-fold increase insensitivity relative to the MWCS testing) provides substantialadditional assurance of the safety of the food products.

Furthermore, as outlined above under “BACKGROUND,” information isfrequently available on a ‘per combo’ basis including, but not limitedto hour of production, vendor source of raw materials, productionemployees present, and operational status of microbial interventionprocess steps. However, when five unrelated combos are included in acombined ‘production lot’ (as in the prior art) it is much harder torationally analyze the available information since there is no way todetermine which combo(s) contained the pathogen (as stated above, priorart re-sampling is not a viable option since it may not yield the sameresult).

By contrast, in this Example I and according to preferred aspects of thepresent invention, any combo-specific information including, but notlimited to hour of production, vendor source of raw materials,production employees present, and operational status of microbialintervention process steps can be brought to bear in the context of the391 individual single-unit production lots yielding a positive sampleresult, thereby providing an effective means not only to tracecontamination, but also to affect remedial measures in view thereof.

TABLE 1 Results of modular wet composite sampling of 62,919 multiplelots and number of Individual Production Unit Lot (IPUL) which testedpositive when MWCS tested positive Total # # 1 2 3 4 5 Location AnalysesPositives IPUL IPULs IPULs IPULs IPULs Est. A, B, C. 62,919 271 180 3918 11 7

Example II According to Particular Aspects of the Present Invention, theInventive Sampling Methods have Substantial Utility in the Context ofReady-to-Eat (RTE) Products (E.g., Meat and Poultry Products)

Under United States Department of Agriculture, Food Safety andInspection Service (USDA FSIS) guidelines, producers of Ready-to-Eat(RTE) meat and poultry products may adapt an ‘end product’ samplingprogram as verification that their product is free of the pathogenListeria monocytogenes. The frequency of end product sampling isselected by the producer/establishment, based on valid samplingstatistics. Typically, samples are collected at random times over aproduction shift (e.g., from ‘cleanup to cleanup’), and from severalproduct lines. The samples are combined to form a composite sample, andenrichment buffer is added to the composite sample to allow Listeriaspp. or L. monocytogenes to grow and multiply under the favorableconditions of enrichment, thereby providing more material that can bedetected by subsequent analysis.

According to particular aspects of the present invention the producer ofRTE meat or poultry products collects samples from individualsingle-unit production lots. Preferably, the producer defines andproduces these single-unit lots in such a fashion as to maximize theinformation available for investigating the nature of a failure. Forexample, possible definitions of an individual single-unit productionlot include, but are limited to all of the product produced by aparticular production line (or produced by a particular production linein a particular period of time), a volume of product such as a pallet ofpacked boxes produced on a particular production line, or a productionarea in the plant, etc.

Individual single-unit production lot samples are collected, to which,in wet enrichment embodiments, enrichment medium/buffer is added.Following incubation under conditions suitable to allow levels of atarget agent or organism that is present to reach detectable levels andbecome uniform or substantially uniform throughout the respectivesample(s), aliquots of the enrichment medium/buffers from individualsingle-unit production lot samples are combined to form a MWCS. The MWCSis then analyzed for the presence or absence of, for example, Listeriaspp. or L. monocytogenes. In the event that a positive result isobtained for the MWCS, enrichment medium/buffer sample from theindividual single-unit production lot samples comprising that MWCS isanalyzed using the same, or augmented testing protocols.

Individual single-unit production lots for which the enrichmentmedium/buffer from the corresponding sample yields a positive result arediverted to cooking, disposal or other safety endpoint. Individualsingle-unit production lots for which the enrichment medium/buffer fromthe corresponding sample yields a negative result are released intocommerce. The latter fraction represents a direct cost savings to theproducer relative to a prior art approaches where a ‘production lot’ isdefined to include multiple single-unit production lots (e.g., multipleproduct lines and an extended period of time (‘cleanup to cleanup’)).

Furthermore, by defining and establishing the individual single-unitproduction lots in such a fashion as to maximize the informationavailable for investigating the nature of a failure, the producer isable to narrow the scope of investigation and apply remediativeresources more effectively and efficiently. For example, where apositive result(s) is associated with one or more specific productionlines, targeted aggressive sanitation is used to resolve the problem.Alternatively, for example, where a positive result(s) is associatedwith one or more specific products, determining whether raw materialsare contaminated, or whether process microbial intervention steps wereoperating properly are effectively and efficiently used.

Example III According to Particular Aspects of the Present Invention,the Inventive Sampling Methods have Substantial Utility for ProvidingCost-Effective and Effective Monitoring Means for Pathogen Levels in NutProducts (e.g., Almond Products) and the Like

A large number of almond products may be produced by using one or moreprocesses including slivering, scalding, blanching, slicing, roasting,and dicing. Anti-microbial treatments may also be applied, such as useof propylene oxide. In practice, these processes may be used in manyvarious combinations, and the output from one process may be the inputfor another. This makes it very difficult to trace finished product allthe way back to raw materials.

As indicated by a recent outbreak, almond products are susceptible tocontamination by Salmonella spp. According to additional aspects, thepresent inventive methods provide a cost-effective means of implementingan effective monitoring program for pathogen levels in nut products(e.g., almond products). In such embodiments, final almond products ofeach type are collected at random times over a production shift. Theproducer defines and establishes individual single-unit production lotsas the amount of product produced during a production shift (from‘cleanup to cleanup’), or as some smaller production unit (e.g., a‘truckload’), provided that one sample was collected from each suchsingle-unit production lot. The single-unit production lot samples arecombined to form a corresponding composite sample, and, in wetenrichment embodiments, enrichment medium/buffer is added to thecomposite sample to allow Salmonella spp. to grow and multiply under thefavorable conditions of enrichment (as described herein above), therebyproviding more material that can be detected by subsequent analysis.

Aliquots of the enrichment medium/buffers from individual single-unitproduction lot samples are combined to form a MWCS. The MWCS areanalyzed for the presence or absence, for example, of Salmonella spp.Where a positive result is obtained for the MWCS, each enrichment bufferfrom the individual single-unit production lot samples comprising thatMWCS is analyzed using the same, or augmented testing protocols.

Individual single-unit production lots for which the enrichment bufferfrom the corresponding sample yields a positive result are returned forapplication of additional microbial intervention steps (e.g., roasting),or are diverted to a safe endpoint (e.g., a cooked product). Individualsingle-unit production lots for which the enrichment buffer from thecorresponding sample yields a negative result are thereby validated andare releasable into commerce. The latter fraction represents a directcost savings to the producer relative to prior art approaches whereeffective testing is not done, and discovery of contamination with apathogen leads to large scale recalls of product.

Furthermore, by defining and establishing the individual productionsingle-unit production lots in such a fashion as to maximize theinformation available for investigating the nature of a failure, theproducer is able to narrow the scope of investigation and applyremediative resources more effectively and efficiently. For example, theprocesses used to produce the almond product(s) yielding positiveresult(s) are rationally analyzed, and associations between the pathogencontamination and one or more of the processes of slivering, scalding,blanching, slicing, roasting, and dicing are determined, and targetedeffective and efficient remedial measures are applied.

Example IV According to Particular Aspects of the Present Invention, theInventive Sampling Methods have Substantial Utility in the Context ofContaminants that Enter a Production Line at a Given Time Point, andthen Clear

Sterility testing of food and pharmaceuticals, or purity testing offermentation processes often involves compositing a number of samples toform a composite test lot. Detection of microbial contamination in thecomposite sample, as discussed in detail herein above, results inrejection of the whole production. Practically speaking, often apinpoint contaminant enters production at a given time point, and thenclears within a few minutes. Aspects of the present invention allow foridentifying the time of entry, and informed elimination of products(operationally linked test lots) flanking the contamination event.

Example V According to Particular Aspects of the Present Invention, theInventive Sampling Methods have Substantial Utility in the Context ofEnvironmental Monitoring (E.g., with Samples of Water, Wastewater,Sludge, Soil, Surface Sponges, Surface Swabs, Condensates, Air orLiquids)

In the context of environmental monitoring, when several samples (water,wastewater, sludge, soil, surface sponges, surface swabs, condensates,air or liquids) are composited and subjected to enrichment, followed bytesting to detect environmental contamination, the current inventionallows for pinpointing the test lots (e.g., operationally-linkedunit(s)) that are positive for the microbe of concern.

1. A method of sampling, testing and validating test lots for microbialcontamination, comprising: a) collecting a plurality of portions fromeach of a plurality of test lots, the test lots each comprising anassemblage of one or more specimens, wherein each test lot is separatelysampled by taking said plurality of portions thereof; b) combining thecollected plurality of portions corresponding to each of the separatetest lots to provide a corresponding set of separate test lot samples,wherein each separate test lot sample is attributed to a particularcorresponding separate test lot; c) incubating the set of separate testlot samples under conditions suitable to allow levels of a targetmicrobial agent or organism that is present in one or more of theseparate test lot samples to reach detectable levels and become uniform,or substantially uniform, throughout the respective one or more separatetest lot samples, to provide a set of separate test lot samples enrichedfor the target microbial agent or organism; d) removing portions of eachenriched separate test lot sample, and combining the removed portions toprovide a modular composite sample; and e) testing of the modularcomposite sample, and individual testing of each of the enrichedseparate test lot samples, using at least one suitable detection assay,for the target microbial agent or organism, wherein when such testing isnegative all of said separate test lot samples are validated, andwherein when such testing is positive with respect to the modularcomposite sample, or with respect to an individual enriched separatetest lot sample, individual separate test lots may nonetheless yet bevalidated by further testing of a portion of respective initiallynegative enriched separate test lot samples using the same or a moresensitive protocol and obtaining a negative test result.
 2. The methodof claim 1, wherein the test lot is selected from the group consistingof an environmental lot comprising one or more operationally-linkedenvironmental specimens, pharmaceutical lot comprising one or moreoperationally-linked pharmaceutical specimens, single-unit product lotcomprising one or more operationally-linked product specimens, andcombinations thereof.
 3. The method of claim 1, wherein the test lot isa single-unit production lot, comprised of operationally-linked productspecimens.
 4. The method of claim 3, wherein product specimens aresynonymous with product portions.
 5. The method of claim 1, furthercomprising incubating the modular composite sample of d), prior totesting in e).
 6. The method of claim 1, wherein incubating eachseparate test lot sample in c) comprises incubating each separate testlot sample with enrichment medium suitable to enrich for the targetmicrobial agent or organism, and wherein removing in d) comprisesremoving a portion of the enrichment medium from each enriched separatetest lot sample and combining the removed portions to provide a modularcomposite sample.
 7. The method of claim 1, wherein testing of themodular composite sample and enriched test lot samples, using at leastone suitable detection assay further comprises a concentration step toconcentrate the microbial agent or organism.
 8. The method of claim 1,wherein testing in e) of the modular composite sample is positive, andwherein the individual separate test lot samples are further tested,such further testing comprises the more sensitive protocol.
 9. Themethod of claim 1, further comprising, forming an additional modularcomposite sample from the set of enriched separate test lot samples ofc) and testing the additional modular composite sample, using the sameor a different suitable assay, to confirm the testing results.
 10. Themethod of claim 1, further comprising, forming an additional modularcomposite sample from an additional enriched separate test lot samplealong with any enriched separate test lot samples of c) that testnegative in e), and testing the additional modular composite sample,using the same or a different suitable assay.
 11. The method of claim 1,further comprising, where testing of the modular composite sample in e)is negative, forming an additional modular composite sample from the setof enriched separate test lot samples of c), and testing the additionalmodular composite sample, using a different suitable assay, to confirmthe negative testing status.
 12. The method of claim 1, wherein the testlot is selected from the lot group consisting of a single combo, singlebin, single pallet, and a production assemblage.
 13. The method of claim1, wherein the number of separate test lots is from 3 to
 10. 14. Themethod of claim 1, further comprising, where testing of the modularcomposite sample is positive, determining which individual separate testlots are positive, application of remedial measures to said separatepositive test lots, and validating by further testing of a portion ofthe respective remediated separate test lots and obtaining a negativetest result.
 15. The method of claim 1, wherein, in a), separatelycollecting a plurality of portions from each of a plurality of test lotscomprises separately collecting from each test lot a number of portionsfrom 5 to
 100. 16. The method of claim 1, wherein, in a) separatelycollecting said plurality of portions from each of said plurality ofseparate test lots comprises separately collecting from each separatetest lot 30 to 60 portions.
 17. The method of claim 1, wherein, in a)separately collecting said plurality of portions from each of saidplurality of separate test lots comprises separately collecting 30portions from each separate test lot.
 18. The method of claim 1,wherein, in a) separately collecting said plurality of portions fromeach of said plurality of separate test lots comprises separatelycollecting from each separate test lot about 60 portions.
 19. A methodof sampling, testing and validating test lots for microbialcontamination, comprising: a) collecting a plurality of portions fromeach of a plurality of single-unit production lots, the single-unitproduction lots each comprising an assemblage of one or more productspecimens, wherein each single-unit production lot is separately sampledby taking said plurality of portions thereof; b) combining the collectedplurality of product portions corresponding to each of the separatesingle-unit production lots to provide a corresponding set of separatesingle-unit production lot samples, wherein each separate single-unitproduction lot sample is attributed to a particular correspondingseparate single-unit production lot; c) incubating the set of separatesingle-unit production lot samples under conditions suitable to allowlevels of a target microbial agent or organism that is present in one ormore of the separate single-unit production lot samples to reachdetectable levels and become uniform, or substantially uniform,throughout the respective one or more separate single-unit productionlot samples, to provide a set of separate single-unit production lotsamples enriched for the target microbial agent or organism; d) removingportions of each enriched separate single-unit production lot sample,and combining the removed portions to provide a modular compositesample; and e) testing of the modular composite sample, and individualtesting of each of the enriched separate test lot samples, using atleast one suitable detection assay, for the target microbial agent ororganism, wherein when such testing is negative all of said separatesingle-unit production lot samples are validated, and wherein when suchtesting is positive with respect to the modular composite sample, orwith respect to an individual separate enriched test lot sample,individual separate single-unit production lots may nonetheless yet bevalidated by further testing of a portion of respective initiallynegative enriched separate single-unit production lot samples using thesame or a more sensitive protocol and obtaining a negative test result.20. The method of claim 1, wherein removing portions in step d) isremoving equal portions, aseptically, so as to preclude contamination ofthe enriched separate test lot samples.
 21. The method of claim 1,wherein the target microbial agent or organism comprises at least oneselected from the group consisting of Listeria, Salmonella andEscherichia coli.
 22. The method of claim 1, wherein the targetmicrobial agent or organism comprises at least one selected from thegroup consisting of Listeria spp., Listeria monocytogenes, Salmonellaspp., Escherichia coli O157:H7, coliforms and fecal coliforms.
 23. Themethod of claim 1, wherein the target microbial agent or organismcomprises a spoilage organism.
 24. The method of claim 1, whereinremoving portions of each enriched separate test lot sample comprisesremoving equal volume portions of each enriched separate test lotsample.
 25. The method of claim 1, wherein testing of the modularcomposite sample, using a suitable detection assay comprises use of atleast one of a DNA-based method, an immunochemistry-based method, and abiosensor-based method.
 26. The method of claim 1, wherein the separatetest lots are production lots of a meat or poultry product, comprisingan assemblage of one or more meat or poultry specimens, respectively.27. The method of claim 1, wherein the separate test lots are productionlots of a fish or seafood product, comprising an assemblage of one ormore fish or seafood specimens, respectively.
 28. The method of claim 1,wherein the separate test lots are production lots of a ready-to-eatproduct.
 29. The method of claim 6, wherein the medium comprises aselective agent that will inhibit a competing non-target microbial agentor organism.
 30. The method of claim 7 wherein the concentration stepcomprises at least one of immunomagnetic bead separation, and affinitychromatography.
 31. The method of claim 8, wherein the more sensitiveprotocol further comprises a concentration step to concentrate themicrobial agent or organism, in combination with the same test protocolused to test the modular composite sample.
 32. The method of claim 19,wherein removing portions in step d) is removing equal portions,aseptically, so as to preclude contamination of the enriched separatesingle-unit production lot samples.
 33. The method of claim 26, whereinthe meat or poultry specimens are meat trim pieces or poultry trimpieces, respectively.
 34. The method of claim 26, wherein the meat isthat of beef, pork, sheep or bison.